Cell culture medium and culture method using the same

ABSTRACT

It is an object of the present invention to provide a cell culture medium capable of enhancing cell growth efficiency without using feeder cells, in particular which does not comprise serum. The present invention provides a cell culture medium which comprises growth arrest-specific 6 (GAS6) and does not comprise serum.

This application is a Divisional of copending application Ser. No.15/039,747 filed on May 26, 2016, which is the U.S. National Phase ofPCT/JP2014/080904, filed Nov. 21, 2014, and which claims priority under35 U.S.C. § 119(a) to Application No. 2013-244758 filed in Japan, onNov. 27, 2013, the entire contents of all of which are expresslyincorporated by reference into the present application.

TECHNICAL FIELD

The present invention relates to a cell culture medium and a method forculturing cells. Also, the present invention relates to feeder cellsthat can be used in the aforementioned culture method, and a method forevaluating a medium for culturing cells.

BACKGROUND ART

As a result of recent studies, the possibility of practical use of humanpluripotent stem cells such as human ES cells (hESC) or human iPS cells(hiPSC) has been increased in the field of regenerative medicine. Thesehuman pluripotent stem cells have an infinite proliferative ability andan ability to differentiate into various cells, and thus, the cells areexpected as a therapeutic means for treating intractable disease,lifestyle-related disease and the like. It has been demonstrated thathuman pluripotent stem cells can be induced to differentiate in vitrointo a variety of cells such as nerve cells, cardiomyocytes, bloodcells, or retinal cells.

Feeder cells act to supply a growth factor for the maintenance cultureof human pluripotent stem cells to stem cells. Thus, human pluripotentstem cells such as hESC or hiPSC have been conventionally culturedmainly on a layer of mouse-derived feeder cells (MEF: mouse embryonicfibroblasts). Activity of performing the maintenance culture of humanpluripotent stem cells has also been reported for various human cells(Non Patent Literatures 1 to 4). However, the method of usingmouse-derived feeder cells has been problematic in terms of safety toliving bodies because the feeder cells are mixed into human pluripotentstem cells. In addition, a culture method of using human-derived feedercells has also been problematic in terms of time-consuming preparationof feeder cells upon culture.

As methods for culturing human pluripotent stem cells without usingfeeder cells such as MEF, a method of previously conditioning a mediumwith MEF (MEF-CM) and a method of chemically immobilizing MEF on amedium have been known (Non Patent Literature 5). Moreover, a method ofusing, as living feeder cells, various human-derived cells such asfibroblasts, placental cells, bone marrow cells or endometrial cells,without using heterologous cells, has also been reported (Non PatentLiterature 6). Furthermore, in order to culture human pluripotent stemcells, a medium containing bovine serum, KNOCKOUT™ SR (Knockout SerumReplacement: an additive that can be used, instead of serum, to cultureES/iPS cells), and the like has been used. However, such a medium oftencontains a protein extracted from bovine serum, and thus, it has beenconcerned about infectious diseases such as bovine spongiformencephalopathy (BSE) or cell contamination caused by virus. There is acase where human-derived serum is used, but since such human-derivedserum has restrictions and limitations on use, it is not suitable forpractical use. Further, it has been known that human pluripotent stemcells can be cultured in the absence of feeder cells by using a mediumfor human pluripotent stem cells containing such serum or a serumreplacement, which has been conditioned with MEF. However, it has beendifficult to identify a factor useful for the growth of humanpluripotent stem cells, which is secreted from MEF, in a mediumcontaining a large amount of serum-derived protein. In order to identifysuch a factor, a receptor protein that changes specifically in human EScells cultured in MEF-CM has also been analyzed (Non Patent Literature7).

The development of a chemically defined medium for carrying out aculture without using MEF has also been promoted, and the problemregarding contamination with bovine serum-derived components has beenalmost avoided (Non Patent Literatures 8 and 9). Using an MEF secretion,the analysis of functional proteins has been carried out (Non PatentLiterature 10). Also, it has been reported that embryonic stem cells canbe cultured without using feeder cells by addition of vitronectin andIGF1 chimeric protein (Non Patent Literature 11). As commerciallyavailable IGF-containing media, mTeSR1 (registered trademark)manufactured by STEM CELL Technologies, STEMPRO (registered trademark)manufactured by Life Technologies, and the like have been known.However, these media have been problematic in that human pluripotentstem cells cannot be stably cultured therein, and in that they exhibitpoor proliferative ability therein.

On the other hand, Patent Literature 1 describes a method for producinga composition comprising a Vitamin K-dependent protein of interest witha substantially lower amount of at least one protein contaminantexpressed endogenous by the host cell in the absence of modification,the method comprising the steps of: a) producing a host cell accordingto the invention; and b) growing the host cell in a growth medium andharvesting the growth medium comprising the Vitamin K-dependent proteinof interest. Patent Literature 1 also describes GAS-6 as an example ofthe vitamin K-dependent protein (paragraphs 0008 and 0035). PatentLiterature 2 describes that cells are cultured in a medium containing10% FCS and GAS6 when a scratch assay is carried out (paragraph 0059).In addition, Patent Literature 3 describes that gas6 is added as aneuron growth enhancer to a medium (paragraph 0235). However, there havebeen no reports regarding addition of GAS6 to a serum-free medium.

Moreover, GAS6 has been clarified to be a ligand for receptor tyrosinekinase (Non Patent Literature 12). The receptor tyrosine kinase includesEGFR, ERBB2, ERBB3, ERBB4, INSR, IGF-1R, IRR, PDGFRα, PDGFRβ, CSF-1R,KIT/SCFR, FLK2/FLT3, VEGRF 1 to 3, FGFR1 to 4, CCK4, TRKA, TRKB, TRKC,MET, RON, EPHA 1 to 8, EPHB 1 to 6, AXL, MER, TYRO3, TIE, TEK, RYK,DDR1, DDR2, RET, ROS, LTK, ALK, ROR1, ROR2, MUSK, AATYK, AATYK2, AATYK3,RTK106, and the like. It has been reported that GAS6 functions as aligand for receptor tyrosine kinase belonging to the TAM family (TYRO3,AXL, and MER) (Non Patent Literature 13).

PRIOR ART LITERATURES Patent Literatures

-   Patent Literature 1: JP Patent Publication (Kohyo) No. 2009-501014 A-   Patent Literature 2: JP Patent Publication (Kohyo) No. 2005-532805 A-   Patent Literature 3: JP Patent Publication (Kokai) No. 2009-77716 A    Non Patent Literatures-   Non Patent Literature 1: Hovatta O, Mikkola M, Gertow K et al., A    culture system using human foreskin fibroblasts as feeder cells    allows production of human embryonic stem cells. Hum Reprod 2003;    18: 1404-1409.-   Non Patent Literature 2: Richards M, Fong C Y, Chan W K et al.,    Human feeders support prolonged undifferentiated growth of human    inner cell masses and embryonic stem cells. Nat Biotechnol 2002; 20:    933-936.-   Non Patent Literature 3: Cheng L, Hammond H, Ye Z et al., Human    adult marrow cells support prolonged expansion of human embryonic    stem cells in culture. Stem Cells 2003; 21: 131-142.-   Non Patent Literature 4: Richards M, Tan S, Fong C Y et al.,    Comparative evaluation of various human feeders for prolonged    undifferentiated growth of human embryonic stem cells. Stem Cells    2003; 21: 546-556.-   Non Patent Literature 5: Yue X-S, Fujishiro M, Nishioka C, Arai T,    Takahashi E, Gong J-S, Akaike T, Ito Y., Feeder cells support the    culture of induced pluripotent stem cells even after chemical    fixation. PLoS ONE 2012; 7: e32707.-   Non Patent Literature 6: Hayato Fukusumi, Yonehiro Kanemura,    Development of feeder cell-free culture technique of human ES/iPS    cells, Igaku no Ayumi (Progress of Medicine), 2011; 239: 1338-1344.-   Non Patent Literature 7: Wang L, Schulz T C, Sherrer E S, Dauphin D    S, Shin S et al., Self-renewal of human embryonic stem cells    requires insulin-like growth factor-1 receptor and ERBB2 receptor    signaling. Blood 2007; 110: 4111-4119.-   Non Patent Literature 8: Veronika A, Peter W A, Stephen B, Nissim B,    Jennifer B et al., Comparison of defined culture systems for feeder    cell free propagation of human embryonic stem cells. In Vitro Cell    Dev Biol Anim. 2010; 46: 247-58.-   Non Patent Literature 9: Chen G, Gulbranson D R, Hou Z, Bolin J M,    Ruotti V, Probasco M D et al., Chemically defined conditions for    human iPSC derivation and culture. Nat Methods. 2011; 8: 424-429.-   Non Patent Literature 10: Chin A C, Fong W J, Goh L T, Philp R, Oh S    K, Choo A B. Identification of proteins from feeder conditioned    medium that support human embryonic stem cells. Journal of    Biotechnology, 2007; 130: 320-8.-   Non Patent Literature 11: Manton K J, Richards S, Van Lonkhuyzen D,    Cormack L, Leavesley D, Upton Z, A chimeric vitronectin: IGF-I    protein supports feeder-cell-free and serum-free culture of human    embryonic stem cells, Stem Cells Dev. 2010, 19(9): 1297-1305-   Non Patent Literature 12: Junichi Shimoda, Takayoshi Hamamoto,    Gas6-deficient mice, Japanese Journal of Thrombosis and Hemostasis,    2001; 12(6): 514-521.-   Non Patent Literature 13: Vyacheslav A K. Axl-dependent signaling: a    clinical update. Clinical science 2012; 122: 361-368.

SUMMARY OF INVENTION Object to be Solved by the Invention

It is an object to be solved by the present invention to provide a cellculture medium capable of enhancing cell growth efficiency without usingfeeder cells, in particular which contains no serum. In addition, it isanother object to be solved by the present invention to provide a methodfor culturing cells using the aforementioned cell culture medium.

Means for Solving the Object

As a result of intensive studies directed towards achieving theaforementioned objects, the present inventor has found that the growthof human pluripotent stem cells can be promoted by adding GAS6 to aserum-free medium used for human pluripotent stem cells, withoutco-culturing the human pluripotent stem cells with feeder cells, therebycompleting the present invention. Specifically, according to the presentinvention, GAS6 has been identified as a factor for promoting the growthof human pluripotent stem cells.

According to the present invention, the following inventions areprovided.

(1) A cell culture medium, which comprises growth arrest-specific 6(GAS6) and does not comprise serum.(2) The cell culture medium according to (1) above, wherein theconcentration of the growth arrest-specific 6 (GAS6) contained in themedium is 2 ng/ml or more and 100 ng/ml or less.(3) The cell culture medium according to (1) or (2) above, which furthercomprises at least one selected from the group consisting of decorin,matrix metalloproteinase-3 (MMP3), osteopontin (OPN), TNF-related weakinducer of apoptosis receptor (TWEAK R), insulin-like growthfactor-binding protein 2 (IGFBP2), galectin 1 (LGALS1), and insulin-likegrowth factor-1 (IGF-1).(4) The cell culture medium according to any one of (1) to (3) above,which does not comprise albumin.(5) The cell culture medium according to any one of (1) to (4) above,wherein the cells are mouse-derived cells or human-derived cells.(6) The cell culture medium according to any one of (1) to (5) above,wherein the cells are pluripotent stem cells.(7) The cell culture medium according to (6) above, wherein thepluripotent stem cells are iPS cells (induced pluripotent stem cells).(8) A cell medium kit, which comprises growth arrest-specific 6 (GAS6)and a basal medium.(9) The cell medium kit according to (8) above, wherein the basal mediumis Essential 8 (registered trademark) medium.(10) The cell medium kit according to (8) or (9) above, which furthercomprises at least one selected from the group consisting of decorin,matrix metalloproteinase-3 (MMP3), osteopontin (OPN), TNF-related weakinducer of apoptosis receptor (TWEAK R), insulin-like growthfactor-binding protein 2 (IGFBP2), galectin 1 (LGALS1), and insulin-likegrowth factor-1 (IGF-1).(11) The cell medium kit according to any one of (8) to (10) above,wherein the cells are mouse-derived cells or human-derived cells.(12) The cell medium kit according to any one of (8) to (11) above,wherein the cells are pluripotent stem cells.(13) The cell medium kit according to (12) above, wherein thepluripotent stem cells are iPS cells or ES cells.(14) A cell culture system, which comprises: (a) the cell culture mediumaccording to any one of (1) to (7) above or the cell medium kitaccording to any one of (8) to (13) above; and (b) a cell cultureapparatus.(15) A method for culturing cells, which comprises culturing cells,using any of the cell culture medium according to any one of (1) to (7)above, the cell medium kit according to any one of (8) to (13) above, orthe cell culture system according to (14) above.(16) The culture method according to (15) above, wherein the cells arecultured in the absence of feeder cells.(17) The culture method according to (15) or (16) above, wherein thecells are cultured on a culture substrate having a surface that has notbeen coated with an extracellular matrix.(18) The culture method according to (15) or (16) above, wherein thecells are cultured on a culture substrate coated with an extracellularmatrix.(19) The culture method according to (17) or (18) above, wherein theextracellular matrix is any one or more selected from the groupconsisting of gelatin, Matrigel that is an isolated basement membranecomponent generated from Engelbreth-Holm-Swarm (EHS) mouse sarcoma,placenta matrix, merosin, tenascin, heparin sulfate, chondroitinsulfate, dermatan sulfate, aggrecan, biglycan, thrombospondin, laminin(laminin-511, laminin-111, and laminin-332), fibronectin, vitronectin,collagen, E-cadherin, decorin, a synthetic peptide, a synthetic polymer,and an extracellular matrix derived from MEF or human serum or deciduamesenchymal cells.(20) The culture method according to any one of (15) to (19) above,wherein the cells are mouse-derived cells or human-derived cells.(21) The culture method according to any one of (15) to (20) above,wherein the cells are pluripotent stem cells.(22) The culture method according to (21) above, wherein the pluripotentstem cells are iPS cells (induced pluripotent stem cells).(23) A method for culturing cells, which comprises culturing cells inthe presence of feeder cells, into which a nucleotide sequence encodingthe amino acid sequence of growth arrest-specific 6 (GAS6) has beenintroduced.(24) The culture method according to (23) above, wherein the feedercells are selected from the group consisting of MEF (mouse embryonicfibroblasts), STO cell line (ATCC Accession No. CRL-1503), fibroblasts,placental cells, bone marrow cells, and endometrial cells.(25) The culture method according to (23) or (24) above, wherein thecells are mouse-derived cells or human-derived cells.(26) The culture method according to any one of (23) to (25) above,wherein the cells are pluripotent stem cells.(27) The culture method according to (26) above, wherein the pluripotentstem cells are iPS cells (induced pluripotent stem cells).(28) Feeder cells, into which a nucleotide sequence encoding the aminoacid sequence of growth arrest-specific 6 (GAS6) has been introduced.(29) The feeder cells according to (28) above, which are selected fromthe group consisting of MEF (mouse embryonic fibroblast), STO cell line(ATCC Accession No. CRL-1503), fibroblasts, placental cells, bone marrowcells, and endometrial cells.(30) A method for evaluating a medium for culturing cells, whichcomprises detecting or quantifying growth arrest-specific 6 (GAS6)contained in the medium.(31) A growth inhibitor for pluripotent stem cells, which comprises ananti-GAS6 antibody or a GAS6 receptor tyrosine kinase inhibitor.(32) The growth inhibitor according to (31) above, wherein

the GAS6 receptor tyrosine kinase inhibitor is one or more selected fromamong:

BMS777607

-   [N-(4-((2-amino-3-chloropyridin-4-yl)oxy)-3-fluorophenyl)-4-ethoxy-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide];

R428

-   [1-(6,7-dihydro-5H-benzo[2,3]cyclohepta[2,4-d]pyridazin-3-yl)-3-N-[(7S)-7-pyrrolidin-1-yl-6,7,8,9-tetrahydro-5H-benzo[7]annulen-3-yl]-1,2,4-triazole-3,5-diamine];    and

UNC569

-   [1-((trans-4-aminocyclohexyl)methyl)-N-butyl-3-(4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-6-amine].    (33) The growth inhibitor according to (31) or (32) above, wherein    the pluripotent stem cells are iPS cells (induced pluripotent stem    cells).    (34) A cell culture medium, which comprises the growth inhibitor    according to any one of (31) to (33) above.    (35) A cell medium kit, which comprises the growth inhibitor    according to any one of (31) to (33) above and a basal medium.    (36) A cell culture system, which comprises: (a) the cell culture    medium according to (34) above or the cell medium kit according    to (35) above; and (b) a cell culture apparatus.    (37) A method for culturing cells, which comprises culturing cells,    using any of the cell culture medium according to (34) above, the    cell medium kit according to (35) above, and the cell culture system    according to (36) above.    (38) A method for culturing pluripotent stem cells, which comprises    culturing pluripotent stem cells under conditions in which the    action of GAS6 is inhibited.    (39) The culture method according to (38) above, wherein the    culturing of pluripotent stem cells under conditions in which the    action of GAS6 is inhibited is a culturing of pluripotent stem cells    in the presence of an anti-GAS6 antibody and/or a GAS6 receptor    tyrosine kinase inhibitor.    (40) The culture method according to (39) above, wherein

the GAS6 receptor tyrosine kinase inhibitor is one or more selected fromamong:

BMS777607

-   [N-(4-((2-amino-3-chloropyridin-4-yl)oxy)-3-fluorophenyl)-4-ethoxy-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide];

R428

-   [1-(6,7-dihydro-5H-benzo[2,3]cyclohepta[2,4-d]pyridazin-3-yl)-3-N-[(7S)-7-pyrrolidin-1-yl-6,7,8,9-tetrahydro-5H-benzo[7]annulen-3-yl]-1,2,4-triazole-3,5-diamine];    and

UNC569

-   [1-((trans-4-aminocyclohexyl)methyl)-N-butyl-3-(4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-6-amine].    (41) The culture method according to (39) above, wherein the    concentration of the anti-GAS6 antibody contained in a medium for    culturing pluripotent stem cells is 10 ng/ml or more and 100 ng/ml    or less.    (42) The culture method according to (40) above, wherein when a    medium for culturing pluripotent stem cells comprises BMS777607, the    concentration of the BMS777607 contained in the medium is 2 ng/ml or    more and 20 ng/ml or less, when a medium for culturing pluripotent    stem cells comprises R428, the concentration of the R428 contained    in the medium is 8 ng/ml or more and 80 ng/ml or less, and when a    medium for culturing pluripotent stem cells comprises UNC569, the    concentration of the UNC569 contained in the medium is 1 ng/ml or    more and 10 ng/ml or less.    (43) The culture method according to any one of (38) to (42) above,    wherein the pluripotent stem cells are iPS cells (induced    pluripotent stem cells).    (44) The culture method according to any one of (38) to (43) above,    wherein the pluripotent stem cells are cultured in a medium, which    does not comprise albumin.    (45) The culture method according to any one of (38) to (44) above,    wherein the pluripotent stem cells are cultured in a medium, which    does not comprise serum.    (46) The culture method according to any one of (38) to (45) above,    wherein the pluripotent stem cells are cultured in a medium    comprising GAS6.    (47) The culture method according to any one of (38) to (46) above,    wherein the pluripotent stem cells are cultured in a medium    conditioned with MEF.

Advantageous Effects of Invention

By culturing cells using the cell culture medium of the presentinvention, it becomes possible to allow cells such as pluripotent stemcells to efficiently grow. According to the cell culture medium of thepresent invention, cells can be cultured without using feeder cells, andthus, the cells can be safely cultured.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows comparative results regarding the growth efficiencies ofhuman iPS cells in a serum-free medium, in a serum-free mediumconditioned with MEF, or in serum-free media to which various types ofprotein factors have been added, which are compared by staining withalkaline phosphatase.

FIG. 2 shows comparative results regarding the growth efficiencies ofhuman iPS cells in a serum-free medium, in a serum-free mediumconditioned with MEF, or in serum-free media to which various types ofprotein factors have been added, which are compared by staining withalkaline phosphatase.

FIG. 3 shows the results obtained by counting the number of human iPScells in a serum-free medium, in a serum-free medium conditioned withMEF, or in serum-free media to which various types of protein factorshave been added, and then comparing the obtained numbers.

FIG. 4 shows comparative results regarding the growth efficiencies ofhuman iPS cells in a serum-free medium, in a serum-free mediumconditioned with MEF, or in serum-free media to which various types ofprotein factors have been added, which are compared by staining withalkaline phosphatase.

FIG. 5 shows the results obtained by counting the number of human iPScells in a serum-free medium, in a serum-free medium conditioned withMEF, or in serum-free media to which various types of protein factorshave been added, and then comparing the obtained numbers.

FIG. 6 shows comparative results regarding the growth efficiencies ofhuman iPS cells, which have been cultured in a serum-free medium or in aserum-free medium conditioned with MEF, without using Matrigel, andwhich have been then compared by staining with alkaline phosphatase.

FIG. 7 shows the results obtained by counting the number of human iPScells in a serum-free medium, in a serum-free medium conditioned withMEF, or in a medium conditioned with MEF, to which an anti-GAS antibodyhas been added, and then comparing the obtained numbers.

FIG. 8 shows the results obtained by counting the number of human iPScells in a serum-free medium, in a serum-free medium conditioned withMEF, or in media conditioned with MEF, to which various types of GAS6receptor tyrosine kinase inhibitors have been added, and then comparingthe obtained numbers.

FIG. 9 shows an outline configuration of individual constitutionalelements of a culture apparatus.

FIG. 10 shows a block diagram of an observation device for an automaticculture apparatus.

FIG. 11 shows a configuration of hardware forming a main part of anautomatic culture apparatus.

FIG. 12 shows a configuration of a cell detection system of a cellculture apparatus.

FIG. 13 shows an outline configuration of individual constitutionalelements of a culture apparatus.

FIG. 14 shows details of an image processing unit.

FIG. 15 shows an example of a cell extraction treatment conducted by animage processing unit.

EMBODIMENTS FOR CARRYING OUT THE INVENTION

Hereinafter, the embodiments of the present invention will be described.

The cell culture medium of the present invention is characterized inthat it contains growth arrest-specific 6 (GAS6) and does not containserum. By adding growth arrest-specific 6 (GAS6) to a medium, the growthof human pluripotent stem cells can be promoted without using serum andfeeder cells. Thereby, human pluripotent stem cells, which are notexposed to either animal cells or a culture solution conditioned withsuch animal cells, can be easily cultured.

The concentration of growth arrest-specific 6 (GAS6) contained in themedium is not particularly limited, as long as cells are able to grow inthe presence of GAS6. Taking into consideration addition effect, ingeneral, the lower limit of the concentration is 0.0001 ng/ml,preferably 0.001 ng/ml, more preferably 0.01 ng/ml, even more preferably0.1 ng/ml, particularly preferably 1 ng/ml, and most preferably 2 ng/ml.Taking into consideration costs, the upper limit of the concentration is10000 ng/ml, preferably 1000 ng/ml, more preferably 500 ng/ml, even morepreferably 400 ng/ml, particularly preferably 300 ng/ml, moreparticularly preferably 200 ng/ml or less, and even more particularlypreferably 100 ng/ml or less. The concentration of GAS6 is generally 1ng/ml or more and 200 ng/ml or less, and particularly preferably 2 ng/mlor more and 100 ng/ml or less.

The cell culture medium of the present invention is characterized inthat cell growth can be promoted and cells can be stably cultured in amedium, which does not comprise serum (namely, a serum-derivedcomponent). The medium that can be used in the present invention can beprepared using a medium used for animal cells as a basal medium. Thebasal medium is not particularly limited, as long as it can be used forthe culture of animal cells. Examples of the basal medium include, butare not particularly limited to, BME medium, BGJb medium, CMRL1066medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDMmedium, Medium 199 medium, Eagle MEM medium, aMEM medium, DMEM medium,Ham medium, RPMI 1640 medium, Fischer's medium, and a mixed mediumthereof. Specific examples include media containing no serum-derivedcomponents, to which insulin and transferrin are added, such asCHO-S-SFM II (manufactured by GIBCO BRL), Hybridoma-SFM (manufactured byGIBCO BRL), eRDF Dry Powdered Media (manufactured by GIBCO BRL),UltraCULTURE™ (manufactured by BioWhittaker), UltraDOMA™ (manufacturedby BioWhittaker), UltraCHO™ (manufactured by BioWhittaker), andUltraMDCK (manufactured by BioWhittaker). Essential 8 (manufactured byLife Technologies), STEMPRO hESC SFM (manufactured by LifeTechnologies), mTeSR1 (manufactured by Veritas), TeSR2 (manufactured byVeritas), and ReproXF (manufactured by ReproCELL), all of which havebeen developed for use in the culture of human pluripotent stem cells,are optimal media. Particularly preferably, DMEM/F12 (1:1) medium orEssential 8 (manufactured by Life Technologies), to which ITS (insulin,transferrin, and selenium), human FGF2, TGF-β1, ascorbyl-2-phosphatemagnesium, and sodium hydrogen carbonate are added, can be used.

To the cell culture medium of the present invention, an MEF secretioncomponent may be further added. Specifically, at least one selected fromthe group consisting of decorin, matrix metalloproteinase-3 (MMP3),osteopontin (OPN), TNF-related weak inducer of apoptosis receptor (TWEAKR), insulin-like growth factor-binding protein 2 (IGFBP2), galectin 1(LGALS1), and insulin-like growth factor-1 (IGF-1) may be added as anMEF secretion component.

Among such MEF secretion components, which can be added to the presentculture medium in addition to growth arrest-specific 6 (GAS6), at leastone selected from the group consisting of decorin, galectin 1 (LGALS1),and insulin-like growth factor-1 (IGF-1) can be preferably added. Forexample, such MEF secretion components and GAS6 can be added incombination of “GAS6 and decorin,” “GAS6 and LGALS1,” “GAS6 and IGF-1,”“GAS6, decorin and LGALS1,” “GAS6, decorin and IGF-1,” “GAS6, LGALS1 andIGF-1,” or “GAS6, decorin, LGALS1 and IGF-1,” etc.

It is to be noted that since decorin, galectin 1 (LGALS1), andinsulin-like growth factor-1 (IGF-1) exhibit the effect of promotingcell growth, even in a case where they are added alone, it is alsopossible to add merely one or more factors selected from the groupconsisting of decorin, galectin 1 (LGALS1) and insulin-like growthfactor-1 (IGF-1) to the cell culture medium.

When the above-described MEF secretion component is added to the cellculture medium, the concentration of each component contained in themedium is not particularly limited, as long as cells can proliferatetherein. Taking into consideration addition effect, in general, thelower limit of the concentration is 0.0001 ng/ml, preferably 0.001ng/ml, more preferably 0.01 ng/ml, even more preferably 0.1 ng/ml,particularly preferably 1 ng/ml, and most preferably 2 ng/ml. Takinginto consideration costs, the upper limit of the concentration is 10000ng/ml, preferably 1000 ng/ml, more preferably 500 ng/ml, even morepreferably 400 ng/ml, particularly preferably 300 ng/ml, moreparticularly preferably 200 ng/ml or less, and even more particularlypreferably 100 ng/ml or less. The concentration of the component isgenerally 1 ng/ml or more and 200 ng/ml or less, and particularlypreferably 2 ng/ml or more and 100 ng/ml or less.

The cell culture medium of the present invention preferably does notcomprise albumin.

The cell culture medium of the present invention may comprise, asappropriate, transferrin, fatty acid, insulin, a collagen precursor, atrace element, 2-mercaptoethanol, 3-thiolglycerol, or an equivalentthereof.

The medium of the present invention may also comprise fatty acid orlipid, amino acid (e.g., non-essential amino acid), vitamin, a growthfactor, cytokine, an antioxidant, 2-mercaptoethanol, pyruvic acid, abuffer agent, inorganic salts, etc. For example, the concentration of2-mercaptoethanol is not limited, as long as it is used in aconcentration suitable for the culture of stem cells. It can be used ina concentration of, for example, approximately 0.05 to 1.0 mM, andpreferably approximately 0.1 to 0.5 mM.

As described above, the cell culture medium of the present invention canbe produced by adding GAS6 to a basal medium. The present cell culturemedium can also be used in the form of a cell medium kit, whichcomprises GAS6 and a basal medium. Specifically, a kit comprising GAS6and a basal medium, wherein the GAS6 and the basal medium are placedseparately, is provided, and when the kit is used, a user adds the GAS6to the basal medium to prepare the cell culture medium of the presentinvention, so that the prepared cell culture medium can be used.

Moreover, according to the present invention, a cell culture systemcomprising (a) the above-described cell culture medium or cell mediumkit and (b) a cell culture apparatus is provided.

The cell culture apparatus is not limited, as long as it is an apparatuscapable of culturing cells using the above-described cell culture mediumor cell medium kit. For instance, the cell culture apparatuses describedin JP Patent Publication (Kokai) No. 2008-92811 A, JP Patent Publication(Kokai) No. 2008-92882 A, and JP Patent Publication (Kokai) No.2010-148391 A (the content described in the aforementioned publicationsis incorporated herein as a part of the disclosure of the presentdescription) can be used.

As an example of the present invention, there can be used a cell cultureapparatus comprising a culture vessel for culturing cells, an imagingdevice for taking an image of the cells in the culture vessel, a lightsource means for applying light to the cells when an image of the cellsis taken, and a moving means for moving the microscope, as described inJP Patent Publication (Kokai) No. 2008-92811 A, wherein the cell cultureapparatus is characterized in that the light source means has aplurality of light sources, and in that the light source correspondingto the position of the microscope is turned on. An example of theconfiguration of the cell culture apparatus is shown in FIG. 9.

A cell culture apparatus 1 is composed of a culture vessel 4 forculturing cells, a microscope 5 for taking an image of the cells, amicroscope driving device 6 for moving the microscope 5, a driver 10 forcontrolling the microscope driving device 6, a switcher 7 for turning onthe power to a specific light source of a plurality of light sources 3established on a light source substrate, based on the positioninformation of the microscope 5, a light source wiring 9 for connectingthe switcher 7 with the light sources 3, a light source substrate 2 onwhich the light sources 3 are aligned, and a controller 8 forcontrolling these components. Moreover, the culture apparatus isconnected with the controller 8, and comprises an input part 20consisting of a trackball or a keyboard. The microscope driving device 6comprises a driving mechanism having a motor and a ball screw mechanism,and is capable of two-dimensionally moving the microscope 5 via the ballscrew mechanism. Furthermore, the ball screw mechanism has a positionsensor which comprises a rotary encoder or the like that detects therotation amount of the ball screw, converts it to the positioninformation of the microscope 5 and detects it. The position sensordetects the rotation amount of the ball screw, so that it cantwo-dimensionally specify the position of the microscope 5. Thisposition information of the microscope 5 is transmitted to thecontroller 8 via the driver 10. The ball screw mechanism comprises aball screw having a spiral groove and a sleeve that moves along thegroove. The microscope 5 is established on the sleeve. By rotating theball screw, the sleeve and the microscope 5 can be linearly moved. Thecell culture apparatus comprises two ball screw mechanisms such thatthey each make a right angle, and as a result, the sleeve and themicroscope 5 can be two-dimensionally moved.

As another example of the present invention, there can be used anautomatic culture apparatus comprising a cell incubator, a camera forphotographing cells in the incubator, a moving means for relativelymoving the position of the camera with respect to the incubator alongthe photographed surface of the incubator, a memory for storing theimage photographed by the camera, a display for displaying the imagestored in the memory, and a control means that is formed to beswitchable between an automatic photographing mode and a manualphotographing mode, as described in JP Patent Publication (Kokai) No.2008-92882 A, wherein the automatic culture apparatus is characterizedin that

the automatic photographing mode has the function of controlling themoving means and allowing the camera to scan with respect to a pluralityof small sections that have been obtained by dividing the photographedsurface by adjusting it to the previously determined visual field of thecamera, then controlling the camera and photographing the surface to bephotographed at a small section unit, then storing the photographedimage in the memory, then reading out the image stored in the memory,and then allowing the display to display the entire image of thephotographed surface, and

(a) the manual photographing mode has the function of controlling themoving means based on the operation command input by the input means andphotographing the local image of the photographed surface at any givenposition by the camera, and at the same time, allowing the display todisplay the thus photographed local image, or

(b) the manual photographing mode has the function of controlling themoving means and moving the position of the camera based on the positionon the display designated by the input means, photographing the localimage at the designated position, and allowing the display to displaythe photographed local image. An example of the configuration of theautomatic culture apparatus is shown in each of FIG. 10 and FIG. 11.

As shown in the perspective view of FIG. 11, the present automaticculture apparatus is configured to establish a circular petri dish 2serving as a cell incubator in an incubator 1, and in the incubator 1, atemperature and a gas concentration such as CO₂ are adjusted, as ispublicly known. The petri dish 2 is established on a bedplate 4supported by a stand 3, and an aperture 5, which does not hinderobservation of the bottom surface of the petri dish 2 from below, isestablished on the bedplate 4. A CCD camera 6 for photographing cells inthe petri dish 2 is established below the aperture 5 on the bedplate 4.The CCD camera 6 comprises an objective lens with a high magnificationcapable of observing cells. It is to be noted that a CMOS camera can beused instead of the CCD camera 6. The CCD camera 6 is mounted on aY-axis actuator 7 for moving the camera in the Y direction indicatedwith the illustrated arrow. The Y-axis actuator 7 is mounted on anX-axis actuator 8 for moving the camera in the X direction with theillustrated arrow. In addition, a light source 54 is disposed at aposition opposing the CCD camera 6 across the petri dish 2, and thelight source 54 is fixed on the CCD camera 6, and it is equipped at thetip of a supporting arm 10. A moving means is configured to relativelymove the positions of the CCD camera 6 and the light source 54 to anygiven positions along the bottom surface of the petri dish 2 by theY-axis actuator 7 and the X-axis actuator 8. The CCD camera 6, theY-axis actuator 7, the X-axis actuator 8 and the light source 54 arecontrolled by a computer 11 such as a personal computer (PC), and anobservation device that is a feature of the present invention iscomposed of these components.

The computer 11 is configured by connecting a computer body 12, adisplay 13, a keyboard 14 and a mouse 15 serving as input means, and thelike. The computer body 12 comprises CPU, memory, I/O, etc., and thus,it is comprises I/O necessary for a general purpose computer.

As shown in FIG. 10, the present apparatus comprises a control unit 20composed of the computer body 12, and the control unit 20 is connectedwith the incubator 1, the CCD camera 6, the Y-axis actuator 7, theX-axis actuator 8, the display 13, the keyboard 14, and the mouse 15. Aninterrupt switch 14 a, a manual switch (X-axis) 14 b, and a manualswitch (Y-axis) 14 c are established on the keyboard 14. Moreover, animage memory 21 for storing the image photographed by the CCD camera 6and a photographing position data memory 22 are connected with thecontrol unit 20. Furthermore, the control unit 20 comprises anon-illustrative reading-out means for reading out the image stored inthe image memory 21 and allowing the display 13 to display it, and acontrol means for controlling the photographing by the CCD camera 6 byswitching it between an automatic photographing mode and a manualphotographing mode.

Details for the operations performed using the present apparatus are asdescribed in paragraphs 0029 to 0053 of JP Patent Publication (Kokai)No. 2008-92882 A.

As a further example of the present invention, as described in JP PatentPublication (Kokai) No. 2010-148391 A, there can be used a celldetection system of a cell culture apparatus comprising an incubatormeans for culturing cells, an image-obtaining means for obtaining theimage of the cells from the incubator means, a focus position adjustingmeans for moving either one of the image-obtaining means and theincubator means and adjusting the focus of the image-obtaining means,and a control means for controlling the image-obtaining means such thatimages can be obtained at a plurality of focus positions of theimage-obtaining means, wherein the cell detection system ischaracterized in that it comprises an image selection means forselecting an image in which the margin of a cell is seen clearly fromthe plurality of images obtained at the plurality of focus positions,and an extraction means for performing a subtraction treatment on afirst image selected by the image selection means, in which the marginof a cell is seen clearly, and a second image obtained at a positiondeviated from the focus position of the first image. An example of theconfiguration of the cell detection system is shown in each of FIG. 12to FIG. 15.

As shown in FIG. 12, a cell culture apparatus 11 has a box structure inwhich the internal portion thereof is interrupted from the externalspace, and in the box structure, the cell culture apparatus comprises anincubator 12 for culturing cells, a CCD camera 13 for photographing thecells in the incubator 12, an image processing unit 14 for processingthe image data obtained by the CCD camera 13, a converter 15 fortransferring the image data to the image processing unit 14, acamera/incubator driving device 16 for moving the CCD camera 13 or theincubator 12, a motor controller 17 for moving the camera/incubatordriving device 16 to any given position, and a light source 18established above the CCD camera 13. The incubator 12 is molded with atransparent material, and the CCD camera 13 is configured to take aphotograph of a transmitted light that has been irradiated from thelight source 18 and has then passed through the incubator 12. In theabove-described configuration, the CCD camera 13 desirably comprises aCCD device with a resolution of approximately 40,000 pixels, and thelight source 18 is not particularly limited, but it is desirably LED inthe present Examples.

FIG. 13 is a view showing an outline of the arrangement of individualconstitutional elements in the culture apparatus 11, and in theillustrative example, an arrangement in a case where the incubator 12 isfixed in the culture apparatus 11 and the CCD camera 13 is moved isshown. As shown in FIG. 13, the light source 18 is equipped in the upperportion of the culture apparatus 11. The incubator 12 is disposed on thelower side of this light source 18. The CCD camera 13 comprising anobjective lens 22 is disposed on the lower side around the center of theincubator 12. The CCD camera 13 is vertically movably equipped in amoving guide 21, and it moves up and down along the moving guide 21according to the drive control by the camera/incubator driving device 16that is allowed to move by the command outputted from a CPU 32, so thatit changes the focus position in the vertical direction and then takes aphotograph of an image of the cells in the incubator 12. An imagingdevice in which the CCD camera 13 is combined with the objective lens 22is desirably configured to be able to take a photograph of a micro arealocated around the bottom surface around the center of the incubator 12,for example, a micro region with a size of about 1.5 mm (depth)×2.0 mm(wide).

FIG. 14 is a view showing details of the image processing unit 14 shownin FIG. 12. The image processing unit 14 is composed of a CPU 32 forperforming arithmetic processing via a data bus 31, a main memory 33temporarily used as a storage area by the CPU 32, an external storagedevice 34 for storing image data or position information, acommunication port 35 for communicating with a motor controller 17, amonitor 36 for displaying the image after cell extraction, and akeyboard 37 for receiving the input of the user. In this imageprocessing unit 14, the image from the CCD camera 13 is incorporatedinto the unit via a converter 15, and various image processing are thencarried out.

FIG. 15 is a flow chart showing an example of a cell extractionprocessing carried out by the image processing unit. A series of stepsshown in FIG. 15 are carried out by previously installed software. Thatis, the steps are carried out by successively drive-controlling theunits shown in FIG. 12 to FIG. 14 by the command outputted from the CPU32, at predetermined time intervals in the cell culture process, forexample, repeatedly at a frequency of once/day. As a timer forrepeatedly carrying out the series of steps shown in FIG. 15 atpredetermined time intervals, an output from a clock generator comprisedin the CPU 32 can be used. Details for the cell extraction treatmentshown in FIG. 15 are as described in paragraphs 0016 to 0028 of JPPatent Publication (Kokai) No. 2010-148391 A.

According to the present invention, a method for culturing cells, whichcomprises culturing cells using any of the above-described cell culturemedium, cell medium kit, or cell culture system of the presentinvention, is provided. Moreover, safe pluripotent stem cells areprovided by culturing cells using any of the cell culture medium, cellmedium kit, and cell culture system of the present invention.

According to the present invention, a method for culturing cells, whichcomprises culturing cells using the above-described cell culture mediumof the present invention, is provided.

The type of cells cultured in the present invention is not particularlylimited, and the cells cultured in the present invention may be eitherstem cells having both pluripotency and self-proliferative ability, ordifferentiated cells. The cells are preferably stem cells, and morepreferably pluripotent stem cells. In the present invention, the“pluripotent stem cells” mean cells that can be cultured in vitro andhave pluripotency by which the cells can differentiate into all cellsconstituting a living body. Specific examples of the pluripotent stemcells include embryonic stem cells (ES cells), fetal gonocyte-derivedpluripotent stem cells (EG cells: Proc Natl Acad Sci USA. 1998, 95:13726-13731), testis-derived pluripotent stem cells (GS cells: Nature.2008, 456: 344-349), and somatic cell-derived induced pluripotent stemcells (induced pluripotent stem cells; iPS cells). Furthermore, animalspecies, from which cells to be cultured are derived, are notparticularly limited, and cells derived from any given mammals and thelike can be used. For example, mouse-derived cells, human-derived cells,or the like can be used. It is to be noted that cells can be culturedunder ordinary cell culture conditions.

In a preferred aspect of the present invention, cells can be cultured inthe absence of feeder cells.

When pluripotent stem cells and the like are cultured without usingfeeder cells, the culture can be carried out using a culture vessel thathas been coated with a culture substrate serving as a scaffold forcells. The culture substrate serving as a scaffold for cells is notparticularly limited, as long as it is used for cell culture. Examplesof the culture substrate include gelatin, Matrigel that is an isolatedbasement membrane component generated from Engelbreth-Holm-Swarm (EHS)mouse sarcoma, placenta matrix, merosin, tenascin, heparin sulfate,chondroitin sulfate, dermatan sulfate, aggrecan, biglycan,thrombospondin, laminin (laminin-511, laminin-111, and laminin-332),fibronectin, vitronectin, collagen, E-cadherin, decorin, a syntheticpeptide, a synthetic polymer, and an extracellular matrix derived fromMEF or human serum or decidua mesenchymal cells.

Moreover, according to another aspect, cells can also be cultured usingthe cell culture medium of the present invention, on a culture substratehaving a surface that has not been coated with the above-describedextracellular matrix.

According to another aspect of the present invention, a method forculturing cells, which comprises culturing cells in the presence offeeder cells, into which a nucleotide sequence encoding the amino acidsequence of growth arrest-specific 6 (GAS6) has been introduced, isprovided. Moreover, according to the present invention, feeder cells,into which a nucleotide sequence encoding the amino acid sequence ofgrowth arrest-specific 6 (GAS6) has been introduced, are provided. Thatis to say, feeder cells, into which a GAS6 gene expression vector hasbeen introduced, can be used in the on-feeder culture of humanpluripotent stem cells, or can also be used in the conditioning of amedium. The type of feeder dells is not limited. Examples of the feedercells include MEF (mouse embryonic fibroblast), the established STO cellline (ATCC Accession No. CRL-1503), mouse-derived cells, such as SNLcells, which are prepared by stably incorporating a neomycin-resistantgene expression vector and an LIF expression vector into STO cells, andhuman-derived cells such as fibroblasts, placental cells, bone marrowcells and endometrial cells.

Moreover, according to the present invention, a method for evaluating amedium for culturing cells, which comprises detecting or quantifyinggrowth arrest-specific 6 (GAS6) contained in the medium, is provided.With regard to the method of detecting and quantifying the GAS6 protein,after the protein has been separated by SDS-PAGE or two-dimensionalelectrophoresis, it can be detected by MS analysis or a Western blotmethod using an anti-GAS6 antibody. Alternatively, it is also possibleto quantifying the protein by an enzyme-linked immunosorbent assay(ELISA).

As described in Examples later, it has been revealed that the growth ofiPS cells can be inhibited by culturing the iPS cells in the presence ofan anti-GAS6 antibody or a GAS6 receptor tyrosine kinase inhibitor.Therefore, according to the present invention, a growth inhibitor forpluripotent stem cells, which comprises an anti-GAS6 antibody or a GAS6receptor tyrosine kinase inhibitor, is provided.

Furthermore, according to the present invention, an anti-GAS6 antibodyfor use to inhibit the growth of pluripotent stem cells and a GAS6receptor tyrosine kinase inhibitor for use to inhibit the growth ofpluripotent stem cells are provided.

Further, according to the present invention, use of the anti-GAS6antibody for the production of a growth inhibitor for pluripotent stemcells and use of the GAS6 receptor tyrosine kinase inhibitor for theproduction of a growth inhibitor for pluripotent stem cells areprovided.

A cell culture medium comprising the growth inhibitor of the presentinvention, a cell medium kit comprising the growth inhibitor of thepresent invention and a basal medium, and a cell culture systemcomprising the cell culture medium or cell medium kit of the presentinvention and a cell culture apparatus are also included in the scope ofthe present invention. Moreover, a method for culturing cells, whichcomprises culturing cells using any of the cell culture medium, cellmedium kit, and cell culture system of the present invention, is alsoincluded in the scope of the present invention. Constitutional elementsother than the growth inhibitor and preferred ranges are as describedabove in the present description.

Furthermore, according to the present invention, a method for culturingpluripotent stem cells, which comprises culturing pluripotent stem cellsunder conditions in which the action of GAS6 is inhibited, is provided.The method for culturing pluripotent stem cells of the present inventioncan be carried out in vitro. By culturing pluripotent stem cells underconditions in which the action of GAS6 is inhibited, the pluripotentstem cells can be cultured in a state in which the growth thereof isinhibited. That is to say, the above-described method for culturingpluripotent stem cells according to the present invention is a methodfor culturing pluripotent stem cells in a state in which the growth ofthe pluripotent stem cells is inhibited, which comprises culturing thepluripotent stem cells under conditions in which the action of GAS6 isinhibited. Specific examples of the pluripotent stem cells are asdescribed above in the present description, and the pluripotent stemcells are preferably iPS cells.

The type of the anti-GAS6 antibody used in the present invention is notparticularly limited, and it may be either a monoclonal antibody or apolyclonal antibody. The anti-GAS6 antibody may also be a commerciallyavailable antibody, and such a commercially available antibody can bepurchased, for example, from R & D Systems, Santa Cruz Biotechnology,etc. The concentration of the anti-GAS6 antibody contained in a mediumfor culturing pluripotent stem cells is not particularly limited. It ispreferably 1 ng/ml or more and 100 ng/ml or less, and more preferably 10ng/ml or more and 100 ng/ml or less.

The type of the GAS6 receptor tyrosine kinase inhibitor used in thepresent invention is not particularly limited. The GAS6 receptortyrosine kinase inhibitor used herein is preferably an inhibitor ofreceptor tyrosine kinases (TYRO3, AXL, and MER) of the TAM family.Specific examples of the GAS6 receptor tyrosine kinase inhibitorinclude: BMS777607[N-(4-((2-amino-3-chloropyridin-4-yl)oxy)-3-fluorophenyl)-4-ethoxy-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide];R428[1-(6,7-dihydro-5H-benzo[2,3]cyclohepta[2,4-d]pyridazin-3-yl)-3-N-[(7S)-7-pyrrolidin-1-yl-6,7,8,9-tetrahydro-5H-benzo[7]annulen-3-yl]-1,2,4-triazole-3,5-diamine];and UNC569[1-((trans-4-aminocyclohexyl)methyl)-N-butyl-3-(4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-6-amine].

In the case of using BMS777607, the concentration of the BMS777607contained in a medium is generally 1 ng/ml or more and 100 ng/ml orless, and preferably 2 ng/ml or more and 20 ng/ml or less. In the caseof using R428, the concentration of the R428 contained in a medium isgenerally 1 ng/ml or more and 500 ng/ml or less, and preferably 8 ng/mlor more and 80 ng/ml or less. In the case of using UNC569, theconcentration of the UNC569 contained in a medium is generally 0.5 ng/mlor more and 100 ng/ml or less, and preferably 1 ng/ml or more and 10ng/ml or less.

The culturing of pluripotent stem cells under conditions in which theaction of GAS6 is inhibited is preferably a culturing of pluripotentstem cells in the presence of an anti-GAS6 antibody and/or a GAS6receptor tyrosine kinase inhibitor.

When pluripotent stem cells are cultured under conditions in which theaction of GAS6 is inhibited, an aspect in which the pluripotent stemcells are cultured in a medium, which does not comprise albumin, anaspect in which the cells are cultured in a medium, which does notcomprise serum, and an aspect in which the cells are cultured in amedium containing GAS6 are preferable, and further, these aspects mayalso be combined with one another.

The present invention will be more specifically described in thefollowing examples. However, these examples are not intended to limitthe scope of the present invention.

Examples (1) Detection of Protein in Conditioned Serum-Free Medium

A conditioned medium (CM) was prepared from a serum-free medium, usingmouse embryonic fibroblasts (MEF cells) that had been inactivated bymitomycin treatment. The mouse embryonic fibroblasts (MEF cells) thathad been inactivated by the mitomycin treatment were inoculated at acell density of approximately 500,000 cells/a dish with a diameter of 60mm in a medium for MEF (DMEM medium containing 10% FBS). The cells werecultured for at least 16 hours, and thereafter, the resulting cells werewashed with PBS(−), and then with an Essential 8 medium (Invitrogen)(hereinafter this serum-free medium is also referred to as an “E8medium”). After that, the medium was replaced with a fresh serum-freemedium of the same type.

After the medium had been replaced with a fresh one, the medium wasincubated for 24 hours in a CO₂ incubator (37° C., 5% CO₂concentration), and it was then recovered, thereby obtaining aconditioned medium.

An unconditioned serum-free medium and a conditioned serum-free mediumwere subjected to comparative analysis using an anti-cytokine antibodyarray. 100 μL of Blocking Buffer (included with a kit) was applied ontoan array of RayBio® Mouse Cytokine Antibody array G-Series 2000(RayBiotech), and it was then incubated at 25° C. for 30 minutes.Thereafter, 150 μL of each medium (dilution magnification: 1.5,approximately 155 μg relative to protein) was applied onto the array,and it was then incubated at 10° C. for 16 hours. Thereafter, theresultant was washed with Wash Buffer 1 (included with the kit, 25° C.,2 minutes×3 times, 25° C., 10 minutes×2 times), and then with WashBuffer II (included with the kit, 25° C., 10 minutes×2 times). Aftercompletion of the washing operations, 70 μL of Biotin-ConjugatedAntibody was applied onto the array, and it was then incubated at 25° C.for 2 hours. After that, the array was washed with Wash Buffer I (25°C., 2 minutes), and then with Wash Buffer II (25° C., 2 minutes×2times). Thereafter, 70 μL of Fluorescent Dye-Conjugated Streptavidin wasapplied onto the array, and it was then incubated at 25° C. for 1.5hours. The array was washed with Wash Buffer I (25° C., 2 minutes), WashBuffer II (25° C., 2 minutes×2 times), Wash Buffer I (25° C., 10minutes×2 times), and ultrapure water (25° C., 1 minute). The washedarray was centrifuged at 1000 rpm at 25° C. for 3 minutes, and was thendried for 20 minutes under light-shielded conditions. Using an arrayscanner GenePix® 4400A (Molecular Devices, LLC), scanning was carriedout, and then, using analysis software Array-Pro Analyzer Ver. 4.5(Media Cybernetics, Inc.), the fluorescence intensity value in each spotwas quantified from the obtained image.

As a result, decorin, matrix metalloproteinase-3 (MMP3), osteopontin(OPN), TWEAK R, insulin-like growth factor-binding protein 2 (IGFBP2),galectin 1 (LGALS1), insulin-like growth factor-1 (IGF-1), and a GAS6protein were detected specifically in the conditioned serum-free medium.

(2) Cytokine Addition Test 1 (2-1) Case of Unconditioned Serum-FreeMedium or Conditioned Serum-Free Medium

Human iPS cells (201B7) used in the experiment were obtained from iPSAcademia Japan, Inc. Human iPS cells were prepared by carrying out amaintenance culture on a plastic culture dish, on which mouse embryonicfibroblasts inactivated by a mitomycin treatment were plated as feedercells. A medium prepared by adding KNOCKOUT™ SR (final concentration:20%), 0.1 mM NEAA (non-essential amino acids), 2 mM L-glutamine, 5 ng/mlhuman basic FGF and 0.1 mM 2-mercaptoethanol to D-MEM/F12 (Sigma D6421)was used. The cells were cultured at 37° C. in a CO₂ incubator.Subculture was carried out every 6 to 7 days, and using a dissociationsolution (collagen solution), a colony of human IFS cells was removedfrom a feeder layer, and about 20 to 50 small masses were then obtainedby a pipetting operation. The obtained small masses were plated on afresh feeder cell layer. Human iPS cells, which had been subjected to amaintenance culture on feeder cells, were removed using a dissociationsolution, and about 20 to 50 small masses were then obtained by apipetting operation. The masses were centrifuged at 300 rpm for 5minutes to recover iPS cells, and MEF was then removed by incubation ofthe cells on a gelatin-coated culture dish for 30 minutes. The resultingcells were divided into 4 portions, and the one portion was theninoculated on a culture dish coated with Matrigel (registered trademark)(BD). As a medium, a serum-free medium (E8 medium) that had not beenconditioned with MEF was used, and it was compared with an E8 mediumthat had been conditioned with MEF.

The results regarding the comparison of proliferative ability are shownin Table 1. Moreover, the results obtained by adding individual factorsto the medium, culturing the mixture for 2 days, and then staining theresultant with alkaline phosphatase, followed by observation, are shownin FIG. 1. It was found that the proliferative efficiency of human iPScells was improved in the conditioned medium, in comparison to in theunconditioned medium.

(2-2)

A factor, namely, decorin, matrix metalloproteinase-3 (MMP3),osteopontin (OPN), TWEAK R, insulin-like growth factor-binding protein 2(IGFBP2), galectin 1 (LGALS1), or insulin-like growth factor-1 (IGF-1),was added in a concentration of 2 ng/ml or 10 ng/ml to a serum-freemedium (E8 medium), and the influence of each factor on the growth ofhuman iPS cells was then examined, as in the case of (2-1) above.

The results regarding the comparison of proliferative ability are shownin Table 1, and the results obtained by staining the cells with alkalinephosphatase and then observing them are shown in FIG. 1 and FIG. 2. Itwas found that decorin, galectin 1 (LGALS1) and insulin-like growthfactor-1 (IGF-1) have the effect of promoting the growth of human iPScells. It has been reported that decorin can be used as a coatingsubstrate for a culture substrate in the culture of human pluripotentstem cells (WO 9920741), and it has also been reported that galectin 1promotes the growth of mouse ES cells (KR2010130677A). IGF-1 factors areadded to a medium such as STEMPRO hESC SFM (manufactured by LifeTechnologies).

(2-3)

GAS6 was added in a concentration of 2 ng/ml or 100 ng/ml to aserum-free medium (E8 medium), and the influence of GAS6 on the growthof human iPS cells was then examined, as in the case of (2-1) above. Theresults regarding the comparison of proliferative ability are shown inTable 1, and the results obtained by staining the cells with alkalinephosphatase and then observing them are shown in FIG. 2. As a result, itcould be confirmed that GAS6 has the effect of promoting the growth ofhuman iPS cells.

(2-4)

All of GAS6, decorin, matrix metalloproteinase-3 (MMP3), osteopontin(OPN), TWEAK R, insulin-like growth factor-binding protein 2 (IGFBP2),galectin 1 (LGALS1), and insulin-like growth factor-1 (IGF-1) were addedin each concentration of 2 ng/ml or 100 ng/ml to a serum-free medium (E8medium), and the influence of these factors on the growth of human iPScells was then examined, as in the case of (2-1) above. The resultsregarding the comparison of proliferative ability with respect to amedium to which only GAS6 was added are shown in Table 1, and theresults obtained by staining the cells with alkaline phosphatase andthen observing them are shown in FIG. 2. A higher effect of promotingthe growth of human iPS cells was found in a medium to which all of theaforementioned 8 factors had been added, than in a medium to which onlyGAS6 had been added.

(3) Cytokine Addition Test 2

As media, the following media were used, and the influence thereof onthe growth of human iPS cells was examined, as in the case of (2-1)above.

Serum-free medium that has not been conditioned with MEF (E8 medium);The above-described E8 medium that has been conditioned with MEF (E8CM);E8 medium, to which any one of decorin (100 ng/ml), matrixmetalloproteinase-3 (MMP3) (2 ng/ml), GAS6 (2 ng/ml), osteopontin (OPN)(100 ng/ml), TWEAK R (2 ng/ml), insulin-like growth factor-bindingprotein 2 (IGFBP2) (2 ng/ml), galectin 1 (LGALS1) (2 ng/ml),insulin-like growth factor-1 (IGF-1) (100 ng/ml) and BA (100 ng/ml) hasbeen added;E8 medium, to which all of decorin (100 ng/ml), matrixmetalloproteinase-3 (MMP3) (2 ng/ml), GAS6 (2 ng/ml), osteopontin (OPN)(100 ng/ml), TWEAK R (2 ng/ml), insulin-like growth factor-bindingprotein 2 (IGFBP2) (2 ng/l), galectin 1 (LGALS1) (2 ng/ml), andinsulin-like growth factor-1 (IGF-1) (100 ng/ml) have been added (E8+8);E8 medium conditioned with MEF, to which all of decorin (100 ng/ml),matrix metalloproteinase-3 (MMP3) (2 ng/ml), GAS6 (2 ng/ml), osteopontin(OPN) (100 ng/ml), TWEAK R (2 ng/ml), insulin-like growth factor-bindingprotein 2 (IGFBP2) (2 ng/ml), galectin 1 (LGALS1) (2 ng/ml), andinsulin-like growth factor-1 (IGF-1) (100 ng/ml) have been added(E8CM+8);E8 medium, to which decorin (100 ng/ml), GAS6 (2 ng/ml), osteopontin(OPN) (100 ng/ml), TWEAK R (2 ng/ml), insulin-like growth factor-bindingprotein 2 (IGFBP2) (2 ng/ml), galectin 1 (LGALS1) (2 ng/ml), andinsulin-like growth factor-1 (IGF-1) (100 ng/ml) have been added(E8+8-MMP3); andE8 medium, to which decorin (100 ng/ml), matrix metalloproteinase-3(MMP3) (2 ng/ml), GAS6 (2 ng/ml), TWEAK R (2 ng/ml), insulin-like growthfactor-binding protein 2 (IGFBP2) (2 ng/ml), galectin 1 (LGALS1) (2ng/ml), and insulin-like growth factor-1 (IGF-1) (100 ng/ml) have beenadded (E8+8-OPN).

The results regarding the comparison of the proliferative ability ofcells are shown in Table 1. In addition, the cells were stained withalkaline phosphatase and the number of cells was then counted, asdescribed in (2-1) above. The number of cells in the case of using an E8medium was defined as 1, and the results regarding the ratio of thenumber of cells in the case of using another medium to that in the caseof using the E8 medium are shown in FIG. 3.

(4) Cytokine Addition Test 3

As media, the following media were used, and the influence thereof onthe growth of human iPS cells was examined, as in the case of (2-1)above.

Serum-free medium that has not been conditioned with MEF (E8 medium);The above-described E8 medium that has been conditioned with MEF (E8CM);E8 medium, to which any one of decorin (100 ng/ml), GAS6 (100 ng/ml),osteopontin (OPN) (100 ng/ml), galectin 1 (LGALS1) (100 ng/ml),insulin-like growth factor-1 (IGF-1) (100 ng/ml) and BSA (100 ng/ml) hasbeen added; andE8 medium, to which all of decorin (100 ng/ml), matrixmetalloproteinase-3 (MMP3) (100 ng/ml), GAS6 (100 ng/ml), osteopontin(OPN) (100 ng/ml), TWEAK R (100 ng/ml), insulin-like growthfactor-binding protein 2 (IGFBP2) (100 ng/ml), galectin 1 (LGALS1) (100ng/ml) and insulin-like growth factor-1 (IGF-1) (100 ng/ml) have beenadded.

The results regarding the comparison of the proliferative ability ofcells are shown in Table 1. The results obtained by staining the cellswith alkaline phosphatase and observing them are shown in FIG. 4. It wasfound that the proliferative efficiency of human iPS cells was improvedin a medium to which GAS6 had been added. In addition, the cells werestained with alkaline phosphatase, and the number of cells was thencounted. The number of cells in the case of using an E8 medium wasdefined as 100, and the results regarding the ratio of the number ofcells in the case of using another medium to that in the case of usingthe E8 medium are shown in FIG. 5.

TABLE 1 Cytokine Cytokine Cytokine addition test 1 addition test 2addition test 3 Concentration (ng/ml) 2 100 2 100 100 Withoutconditioning − − − With conditioning ++ ++ + Decorin + ++ + + MMP3 + + +OPN + + ± ± TWEAK R + + + IGFBP2 ++ ++ + Galectin 1 ++ ++ + + IGF-1 ++++ + + GAS6 + + + + Total (8 factors) +++ +++ + + +

(5) As to Matrigel

Using a culture dish that had not been coated with Matrigel (registeredtrademark) (BD), instead of using a culture dish that had been coatedwith Matrigel (registered trademark) (BD), and using an unconditionedserum-free medium or a conditioned serum-free medium, as in the case of(2-1) above, a comparison was made in terms of the proliferative abilityof human iPS cells. The results obtained by staining the cells withalkaline phosphatase and then observing them are shown in FIG. 6. In thecase of using a conditioned medium, the cells could proliferate even inthe case of using a culture dish that had not coated with Matrigel.

(6) As to Anti-GAS6 Antibody

Using a culture dish that had been coated with Matrigel (registeredtrademark) (BD), 100 ng/ml anti-GAS6 antibody (R & D Systems) was addedto a serum-free medium (E8 medium) conditioned with MEF, and theinfluence of the antibody on the growth of human iPS cells was thenexamined, as in the case of (2-1) above. The cells were stained withalkaline phosphatase, and the number of cells was then counted. Thenumber of cells in the case of using a medium conditioned with MEF wasdefined as 100, and the results regarding the ratio of the number ofcells in the case of using another medium to that in the case of usingthe medium conditioned with MEF are shown in FIG. 7. By adding theanti-GAS6 antibody, the proliferative ability of cells in the mediumconditioned with MEF was reduced.

(7) As to Anti-GAS6 Antibody and GAS6 Receptor Tyrosine Kinase Inhibitor

Using a culture dish that had been coated with Matrigel (registeredtrademark) (BD), 10 ng/ml or 100 ng/ml anti-GAS6 antibody (R & DSystems), or various types of GAS6 receptor tyrosine kinase inhibitors,namely, BMS777607 (Santa Cruz Biotechnology) (2 ng/ml or 20 ng/ml), R428(Synkinase) (8 ng/ml or 80 ng/ml), and UNC569 (Calbiochem) (1 ng/ml or10 ng/ml) were each added to a serum-free medium conditioned with MEF(E8 medium), and the influence thereof on the growth of human iPS cellswas then examined, as in the case of (2-1) above. These factors wereeach dissolved in DMSO, and the obtained solution was then subjected toa filter sterilization treatment using a 0.22-μm filter. The thustreated solution was added to the medium in an amount of 1/1000 theamount of the medium. The cells were stained with alkaline phosphatase,and the number of cells was then counted. The number of cells in thecase of using a medium conditioned with MEF was defined as 100, and theresults regarding the ratio of the number of cells in the case of usinganother medium to that in the case of using the medium conditioned withMEF are shown in FIG. 8. By adding an anti-GAS6 antibody or varioustypes of GAS6 receptor tyrosine kinase inhibitors, the proliferativeability of the cells in the medium conditioned with MEF was reduced.

1. A cell culture medium, which comprises growth arrest-specific 6(GAS6), a basal medium, and at least one selected from the groupconsisting of decorin, matrix metalloproteinase-3 (MMP3), osteopontin(OPN), TNF-related weak inducer of apoptosis receptor (TWEAK R),insulin-like growth factor-binding protein 2 (IGFBP2), galectin 1(LGALS1), and insulin-like growth factor-1 (IGF-1), wherein the cellculture medium does not comprise serum, and the concentration of theGAS6 contained in the cell culture medium is between 2 ng/ml and 100ng/ml.
 2. The cell culture medium according to claim 1, wherein the cellculture medium supports the growth of iPS cells (induced pluripotentstem cells).
 3. The cell culture medium according to claim 1, whereinthe basal medium is BME medium, BGJb medium, CMRL1066 medium, GlasgowMEM medium, Improved MEM Zinc Option medium, DMEM medium, Medium 199medium, Eagle MEM medium, aMEM medium, DMEM medium, Ham medium, RPMI1640 medium, Fischer's medium, or a mixed medium thereof.
 4. The cellculture medium according to claim 1, wherein the basal medium isCHO-S-SFM Hybridoma-SFM, eRDF Dry Powdered Media, UltraCULTURE™,UltraDOMA™, UltraCHO™, and UltraMDCK™, Essential 8, STEMPRO hESC SFM,mTeSR1, TeSR2, or ReproXF.
 5. The cell culture medium according to claim1, comprising decorin, galectin 1 (LGALS1), and insulin-like growthfactor-1 (IGF-1).
 6. The cell culture medium according to claim 1,excluding albumin.
 7. The cell culture medium according to claim 1,excluding a serum-derived component.
 8. The cell culture mediumaccording to claim 1, which is a liquid cell culture medium.
 9. The cellculture medium according to claim 2, which is a liquid cell culturemedium.